Gene encoding a multidrug-resistance human p-glycoprotein homologue on chromosome 7p15-21 and uses thereof

ABSTRACT

The invention relates to an MDR family P-glycoprotein located on human chromosome 7p15-21, polynucleotide sequences encoding this P-glycoprotein and fragments thereof. This gene is utilized in methods for assessing cancer cell s susceptibility to therapies directed against multidrug resistance, and for the design of diagnostic and therapeutic methods relating to cancer multidrug resistance. The invention also relates to methods for determining whether a test compound may inhibit multidrug resistance.

RELATED APPLICATIONS

This application is a continuation of and claims the benefit under 35U.S.C. § 120 of U.S. application Ser. No. 10/952,328, entitled “GeneEncoding A Multidrug-Resistance Human P-Glycoprotein Homologue onChromosome 7P15-2 and Uses Thereof,” filed on Sep. 29, 2004, nowallowed, which is a divisional of U.S. application Ser. No. 09/873,409,filed Jun. 5, 2001, now U.S. Pat. No. 6,846,883 which claims priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No.60/208,913, filed on Jun. 5, 2000, each of which are herein incorporatedby reference in their entirety.

BACKGROUND OF INVENTION

1. Field of Invention

The invention relates to genetic sequences encoding proteins whichexhibit structural and functional features characteristic ofP-glycoprotein family members associated with cancer multidrugresistance, immune regulatory functions, and unique functions in humanpluripotent stem cells and other tissue progenitor cells. The inventionencompasses substantially pure proteins, therapeutic treatments anddiagnostic uses related to these proteins.

P-glycoprotein, an adenosine triphosphate (ATP)-dependent drug effluxpump, is overexpressed in multidrug-resistant (MDR) tumor cells, Itreduces the intracellular concentration of cytotoxic xenobiotics,thereby decreasing the effectiveness of many cancer chemotherapeuticregimens. P-glycoprotein belongs to the ABC (ATP-binding cassette)superfamily of active transporters, and is encoded by a multigene familyin higher eukaryotes. Mammalian P-glycoprotein family members can bedivided into three classes. Class I and class II P-glycoproteins confermultidrug resistance whereas class III proteins do not.

In humans, P-glycoprotein is encoded by two linked genes (“MDR1” and“MDR3”) on chromosome 7g21.1. MDR3 functions as a lipid translocase andmutations in this gene are associated with familial intrahepaticcholestasis. MDR1 confers drug resistance on certain cancer cells. Inaddition to being overexpressed in cancer cells, MDRI P-glycoprotein iswidely expressed in normal, predominantly secretory and absorptive humantissues, where it functions in diverse physiologic processes includingcellular differentiation, cell proliferation and cell survival. In thesenormal cell types, P-glycoprotein functions in the transmembrane releaseor uptake of xenobiotics and certain therapeutic drugs, small peptidemolecules, certain steroid compounds, and phospholipids.

P-glycoprotein is also expressed by lymphoid cell populations from humanbone marrow and the peripheral blood. Specifically, P-glycoprotein hasbeen shown to be expressed on the membrane of pluripotent stem cells,monocytes, dendritic cells, CD4+ and CD8+ T lymphocytes, natural killercells, and B lymphocytes. In immune cells, P-glycoprotein functions inthe transport of cytokines and other small molecules, which are criticalfor physiologic immune responses to occur. Specific blockade ofP-glycoprotein can suppress the immune response to alloantigen andnominal antigen. However, a degree of redundancy exists forP-glycoprotein function in these cell types, pointing to the existenceof additional, hitherto unidentified related molecules.

Pluripotent stem cells and other tissue progenitor cells also possess aunique P-glycoprotein-like activity, characterized by decreasedintracellular accumulation of fluorescent dyes, which allows for thespecific isolation of these cell types for therapeutic uses. However, itis thought that this function is not mediated by MDR1 P-glycoprotein,but rather by a related, as yet unidentified, P-glycoprotein familymember.

Despite the irrefutable role of MDR1 P-glycoprotein in cancer multidrugresistance, attempts to improve chemotherapy by inhibiting this proteinhave met with only limited success. Thus, it may be inferred that thereare homologous proteins that, like MDR1, are able to make cellsresistant to therapeutic agents. In addition, it may be inferred thatMDR1 homologous proteins serve P-glycoprotein-like functions inphysiologic human tissues, in particular in cells of the immune system,pluripotent stem cells and tissue progenitor cells, where eitherredundancy exists for MDR1 P-glycoprotein function, or where MDR1P-glycoprotein is known to not promote the observedP-glycoprotein-associated activity.

SUMMARY OF INVENTION

The invention is directed to a new member of the human P-glycoproteinfamily of genes located on chromosome 7p15-2, encoding proteins whichconfer the multidrug resistant phenotype to tumor cells and/or servecritical physiologic functions in normal human tissues.

An examination of the structure of the new gene indicates that itencodes two semiautonomous homologous halves, each with their owntransmembrane and ATP-binding domains. By alternative splicing anddifferential gene expression and/or posttranscriptional andposttranslational modifications, the new P-glycoprotein gene can encodeseveral distinct P-glycoproteins:

The protein of SEQ ID NO:1 (amino acids 1-659) is encoded by 14 exons(SEQ ID NO:9) of human genomic DNA from clone AC005060 on chromosome7p15-21 and is made up of 5 transmembrane domains and one ATP-bindingdomain.

The protein of SEQ ID NO:2 (amino acids 1-812) is encoded by 19 exons(SEQ ID NO:10) of human genomic DNA from the contiguous clones AC002486and AC005060 (AC002486 is the clone sequenced to the left of cloneAC005060) on chromosome 7p15-21 and is made up of 5 transmembranedomains and two ATP-binding domains, of which the first is located onthe N-terminal side of transmembrane domain #1, and the second on theC-terminal side of transmembrane domain #5 of the protein, on theopposite side of the plasma membrane. The protein of SEQ ID NO:2 canalso be expressed as a result of transsplicing of the mRNA (SEQ ID NO:9) encoding the protein of SEQ ID NO:1 and mRNA (SEQ ID NO: 11) encodingthe protein of SEQ ID NO:3 described hereafter. In addition, the proteinof SEQ ID NO:2 may be expressed as a result of posttranslationalprocessing of the proteins of SEQ ID NO:1 and NO:3.

The protein of SEQ ID NO:3 (amino acids 1-131) is encoded by 6 exons(SEQ ID NO:11) of human genomic DNA from clone AC002486 on chromosome7p15-21 and is made up of one ATP-binding domain and no transmembranedomains.

The protein of SEQ ID NO:4 (amino acids 1-1058) is encoded by exons (SEQID NO:12) of human genomic DNA from the contiguous clones AC002486 andAC005060 on chromosome 7p15-21 and is made up of 8 transmembrane domainsand two ATP-binding domains, of which the first is located betweentransmembrane domains #3 and #4, and the second on the C-terminal sideof transmembrane domains #8; on the opposite side of the plasmamembrane.

The protein of SEQ ID NO:5 (amino acids 1-1222) is encoded by 23 exons(SEQ ID NO:13) of human genomic DNA from the contiguous clones AC002486and AC005060. on chromosome 7p15-21 and is made up of 12 transmembranedomains and two ATP-binding domains, of which the first is locatedbetween transmembrane domains #7 and #8, and the second on theC-terminal side of transmembrane domain # 12, on the opposite side ofthe plasma membrane.

The protein of SEQ ID NO:6 (amino acids 1-1195) is encoded by 24 exons(SEQ ID NO:14) of human genomic DNA from the contiguous clones AC002486and AC005060 on chromosome 7p15-21 and is made up of 11 transmembranedomains and two ATP-binding domains, of which the first is locatedbetween transmembrane domains #6 and #7, and the second on theC-terminal side of transmembrane domain #11, on the opposite side of theplasma membrane.

The protein of SEQ ID NO:7 (amino acids 1-541) is encoded by 10 exons(SEQ ID NO:15) of human genomic DNA from clone AC002486 on chromosome7p15-21 and is made up of 7 transmembrane domains and one ATP-bindingdomain on the C-terminal side of transmembrane domain #7P.

The protein of SEQ ID NO:8 (amino acids 1-514) is encoded by 11 exons(SEQ ID NO:16) of human genomic DNA from clone AC002486 on chromosome7p15-21 and is made up of 6 transmembrane domains and one ATP-bindingdomain on the C-terminal side of transmembrane domain #6.

Cancer multidrug resistance may result from the expression of any of theproteins of SEQ ID NO:1, NO:2, NO:3, NO:4, NO:5, NO:6 NO:7 and NO:8. Theproteins encoded by the 7p15-21 P-glycoprotein gene of the presentinvention may be used as markers for identifying cells likely to displaymultidrug resistance and can serve as targets in the design of newtherapies for cancer patients. It will be understood that, except asotherwise indicated, reference to the P-glycoprotein of the presentinvention also includes any of the proteins of SEQ ID NO:1, NO:2, NO:3,NO:4, NO:5, NO:6, NO:7 and NO:8 as well.

The 7p15-21 P-glycoprotein confers chemoresistance to multiplechemotherapeutic agents, including cisplatinum, by mediating cellulardrug efflux. Hence, specific blockade of this efflux function, forexample by means of specific monoclonal antibody inhibition, can enhanceintracellular drug accumulation and, as a result, drug toxicity andtumor cell killing. In addition, since 7p15-21 P-glycoprotein isfunctional in tumor cell proliferation, tumor growth can betherapeutically inhibited by administration of blocking specificmonoclonal antibodies, even in the absence of concurrentchemotherapeutic agents. Among the proteins encoded by the 7p15-21P-glycoprotein gene, the proteins of SEQ ID NO: 1, NO:2, NO:3, NO:4,NO:5 and NO:6 are distinct from the proteins of SEQ ID NO:7 and NO:8 inthat they are selectively expressed in certain cancer cells but not innon-cancerous normal tissues. Furthermore, the proteins of SEQ ID NO:1,NO:2, NO:3, NO:4, NO:5 and NO:6 are expressed preferentially in thosecancers which exhibit the highest degrees of chemoresistance tochemotherapeutic drugs, such as for example human malignant melanoma.Because of their selective expression in certain cancers but not innormal tissues, the proteins of SEQ ID NO:1, NO:2, NO:3, NO:4, NO:5 andNO:6 can be therapeutically targeted not only via inhibition ofcytotoxic drug efflux or inhibition of tumor proliferation by specificmonoclonal antibodies, but also by additional means, includingtumor-specific cell killing mediated by cell toxin-conjugated specificmonoclonal antibodies, or by therapeutic administration to afflictedpatients of tumor antigen-specific vaccine preparations.

The proteins of SEQ ID NO:7 and NO:8 encoded by the 7p15-21 gene canalso be expressed in certain non-cancerous normal human tissues. Theinvention thus provides for additional uses as relating to the functionof these select proteins in physiologic tissues. Among those normaltissues, the proteins of SEQ ID NO:7 and SEQ ID NO:8 are preferentiallyexpressed at high levels in pluripotent stem cells and other tissueprogenitor cells, where they function in the transmembrane transport ofxenobiotics and other small molecules. The invention provides thus formeans to specifically detect and enrich these stem cells, and progenitorcells from cell mixtures and preparations in which they are contained,by detection of the cells with labeled specific monoclonal antibodies.

The proteins of SEQ ID NO:7 and NO:8 are also expressed to a certaindegree in most other normal human tissues, including in cells of theimmune system such as T cells, monocytes and differentiated antigenpresenting cells, where they function in the efflux of cytokines and theuptake of small molecules including peptides and antigen, thus serving acritical role for the integrity of normal immune responses. When thesefunctions are inhibited, for example by specific monoclonal antibodyblockade, the normal immune response can be modulated, which can beutilized in the prevention and/or the therapy of allograft rejection inclinical organ transplantation, and also in various autoimmune diseasessuch as rheumatoid arthritis and multiple sclerosis. In addition, whenexpressed in human immune cells and other human tissues such as theendothelium of the blood-brain barrier and the epithelia of thegastrointestinal tract and the kidney, blockade of the protein canfurthermore be therapeutically employed to selectively alter the uptakeand secretion, and hence the pharmacological distribution,pharmacokinetics and therapeutic efficacy of those, exogenouslyadministered therapeutic drugs which are substrates of said proteins.

In a first aspect, the invention is directed to substantially pureproteins consisting essentially of the amino acid sequence of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7 or SEQ ID NO:8. The term “consisting essentially of” ismeant to encompass proteins having exactly the same amino acidsequences, as well as proteins with insubstantially different sequences,as evidenced by their possessing the same basic functional properties. A“substantially purified” isoform is one that has been separated fromother accompanying biological components and will typically comprise atleast 85% of a sample, with greater percentages being preferred. Manymeans are available for assessing the purity of a protein within asample, including analysis by polyacrylamide gel electrophoresis,chromatography and analytical centrifugation. A preferred method forassessing purity is by Western blotting using an antibody directedagainst epitopes of the 7p15-21 P-glycoprotein of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 orSEQ ID NO:8. The invention also encompasses “MDR peptides” which aredefined herein as consisting of a sequence element of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7or SEQ ID NO:8 of at least 10 and preferably at least 15 or 20 residues.These may be used in the generation of antibodies. It is stipulated thatan MDR peptide cannot have a sequence that is the same as any set of 10to 15 contiguous residues in the sequence LSGGQKQRIAIARAL (SEQ IDNO:17). These proteins and MDR peptides may also be administeredtherapeutically to cancer patients afflicted with 7p15-21 P-glycoproteinexpressing tumors, as a tumor vaccine to elicit an endogenous immuneresponse directed against these tumors, to result in tumor-specific cellkilling.

In another embodiment, the invention is directed to an antibody made bya process comprising the step of administering to an animal host aprotein encoded by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or an MDR peptideas described above. The protein or peptide should be administered to theanimal at a dosage sufficient to induce antibody formation. Antibodiesmay be monoclonal or polyclonal. In the latter case, antibodies arepreferably produced by injecting a pharmaceutically acceptablepreparation into a mouse, followed by fusing mouse spleen cells withmyeloma cells using techniques known in the art. The antibodies obtainedshould bind selectively to the proteins of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ IDNO:8. Selective binding, in this context, means that an antibody has atleast a 100-fold greater affinity for one or more of these proteins thanfor any other protein normally found in human cells.

The invention is also directed to a substantially pure polynucleotideconsisting essentially of a nucleotide sequence encoding the proteins ofSEQ ID NO:1, SEQ ID NO;2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7 or SEQ ID NO:8, or an MDR peptide. Preferably, thepolynucleotide consists essentially of the nucleotide sequence of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15 or SEQ ID NO: 16. The invention includes expressionvectors comprising a distinct coding clement consisting of thesepolynucleotides; and host cells transformed with such vectors. A“distinct coding element” refers to the portion of an expression vectorresponsible for determining the amino acid sequence of an expressedprotein. The invention comprises all such elements producing proteinscorresponding to the amino acid sequences shown in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 orSEQ ID NO:8, as well as other proteins having substantially the samestructure and function.

The invention includes recombinant protein made by host cellstransformed by an expression vector as discussed above. The recombinantprotein may be isolated using standard techniques, including affinitychromatography with antibodies against epitopes of 7p15-21P-glycoprotein. Preferably, the polynucleotide used in vectors forexpressing such a recombinant P-glycoprotein consists essentially of thenucleotide sequences of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.Oligonucleotides complementary to SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQID NO: 16 and at least 15 nucleotides in length maybe used as antisenseinhibitors. These may be administered to patients undergoing cancerchemotherapy to increase the effectiveness of cytotoxic drugs. The invivo transfection of cells has been known for many years and may beaccomplished using viral vectors (see e.g., U.S. Pat. No. 6,020,191);liposomes (see e.g., Nicolau, Meth. Enzymol 149:157-176 (1987)); DNAcomplexed to agents that facilitate cellular uptake (see e.g., U.S. Pat.No. 5,264,618; WO 98/14431); or even by simply injecting naked DNA (seee.g., U.S. Pat. No. 5,693,622). Any of these procedures may be used todeliver the antisense oiligonucleotides of the present invention.

The invention is also directed to a method for determining whether acancer cell will respond to therapies aimed at reversing multidrugresistance by measuring the expression of the genes encoding theproteins of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. This method may be usedto detect the existence of the multidrug resistant phenotype in cancercells or to track the development of multidrug resistance over time bymonitoring changes in gene expression in cultured cells.

In another embodiment, the invention provides for a method ofdetermining whether a test compound inhibits multidrug resistance incells caused by a gene encoding proteins of SEQ ID: NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQID NO:8. This method comprises expressing a gene encoding one or more ofthese polypeptides in cells that are otherwise not multidrug resistantand exposing these cells to one or more cytotoxic drugs in the presenceof a test compound. Cellular survival is measured after exposure and theresults obtained are compared with those from incubations carried out inessentially the same manner but in the absence of the test compound. Itis concluded that the test compound inhibits multidrug resistance ifcellular survival is decreased to a significant extent in incubationscarried out in the presence of the test compound relative to that seenin its absence.

DETAILED DESCRIPTION

The invention is directed to a novel member of the P-glycoprotein familyof drug resistance related proteins, to genetic sequences encoding thisprotein, to methods of determining whether a cancer cell will respond totherapies aimed at reversing P-glycoprotein mediated drug resistance,and to a method of screening test compounds for their ability to inhibitmultidrug resistance. The novel P-glycoprotein gene can encode theproteins of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

It will be understood that the invention encompasses not only sequencesidentical to those shown but also sequences that are essentially thesame as evidenced by their retaining the same basic structural andfunctional characteristics. For example, techniques such as sitedirected mutagenesis may be used to introduce variations into aprotein's structure. Variations in P-glycoprotein introduced by this orother similar methods are encompassed by the invention provided that theresulting protein retains its basic biological properties, particularlywith respect to the inducement of multidrug resistance in mammaliancells.

DNA sequences encoding the proteins of the invention may be obtainedfrom any tissue or cellular source in which they are expressed. Forexample, cultured cell lines may be engineered to express theP-glycoprotein gene using recombinant techniques or by continuousexposure to chemotherapeutic agents. Alternatively, sequences may beisolated from primary cells obtained from tumors.

Many methods are available for isolating DNA sequences and may beadapted for the isolation of the chromosome 7p15-21 (hereinafter“chromosome 7p”) P-glycoprotein gene (see, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborPress (1989)). For example, one method is to screen a cDNA library thathas been prepared by reverse transcribing mRNA isolated from tissues orcells that express the gene. The library may be prepared from, forexample, human melanocyte or testis tissue and probes for screening maybe synthesized based upon the sequences shown in the Sequence Listing.The probes are preferably at least 14 nucleotides long and are optimallyselected from a region believed to be unique to the chromosome 7 pP-glycoprotein gene.

As an alternative, amplification of a desired sequence may be achievedby the polymerase chain reaction (“PCR”) of reverse transcribed RNA.Primers for PCR may be constructed using the sequences shown in SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 or SEQ ID NO:16, and confirmation of the presence ofchromosome 7p P-glycoprotein cDNA may be obtained by the sequencing ofamplification products.

Expression of recombinant protein may be induced in a host cell bytransforming it with an appropriate expression vector. The vector shouldcontain transcriptional and translational signals recognizable by thehost together with the desired structural sequence, preferably in doublestranded form, in an operable linkage. For example, the P-glycoproteinDNA sequence should be positioned such that regulatory sequences presentin the vector control the synthesis of mRNA and protein having thedesired sequence is produced.

Preferably, nucleic acid encoding the P-glycoprotein of the invention isexpressed in eukaryotic cells, especially mammalian cells. Such cellsare capable of promoting post-translational modifications necessary toensure that the recombinant protein is structurally and functionally thesame as the protein isolated from, for example, multidrug resistanttumor cells. Examples of mammalian cells known to provide properpost-translational modification of cloned proteins include, inter alia,NIH-3T3 cells, CHO cells, HeLA cells, LM(tk-) cells, and the like.Eukaryotic promoters known to control recombinant gene expression arepreferably utilized to drive transcription of chromosome 7pP-glycoprotein DNA, and may include that of the mouse metallothionein Igene, the TK promoter of Herpes virus, the CMV early promoter and theSV40 early promoter. Transcription may also be directed by prokaryoticpromoters, such as those capable of recognizing T4 polymerase, the P_(R)and P_(L) promoters of bacteriophage lambda, and the trp, recA, heatshock and lacZ promoters of E. coli.

Expression vectors may be introduced into host cells by methods such ascalcium phosphate precipitation, microinjection, electroporation orviral transfer and cells expressing the recombinant protein sequence canbe selected by techniques known in the art. Confirmation of expressionmay be obtained by PCR amplification of P-glycoprotein sequences usingprimers selected from the sequences shown in SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 orSEQ ID NO:16.

Recombinant protein may be purified using standard techniques well knownin the art Such techniques may include filtration, precipitation,chromatography and electrophoretic methods. Purity can be assessed byperforming electrophoresis on a polyacrylamide gel and visualizingproteins using standard staining methodology. Western blotting also maybe performed using an antibody to chromosome 7p P-glycoprotein.

The invention is also directed to antibodies raised against thechromosome 7p P-glycoprotein. The process for producing such antibodiesmay involve either injecting the 7p P-glycoprotein itself into anappropriate animal or injecting short antigenic peptides made tocorrespond to different regions of the protein. These peptides should beat least 5 amino acids in length and should, preferably, be selectedfrom regions believed to be unique to the 7p P-glycoprotein. Methods forgenerating and detecting antibodies are well known in the art, and aretaught by such references as: Harlow, et al., Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory, NY (1988); Klein, Immunology: TheScience of Self-Nonself Discrimination, (1982); Kennett et al.,Monoclonal Antibodies and Hybridomas: A New Dimension in BiologicalAnalyses, (1980); and Campbell, “Monoclonal Antibody Technology”, inLaboratory Techniques in Biochemistry and Molecular Biology, (1984).

The term “antibody”, as used herein, is meant to include intactmolecules as well as fragments that retain their ability to bindantigen, such as Fab and F(ab′)₂ fragments. The term “antibody” is alsodefined herein as relating to both monoclonal antibodies and polyclonalantibodies. Polyclonal antibodies are derived from the sera of animalsimmunized with a chromosome 7p P-glycoprotein antigen. Monoclonalantibodies to the protein can be prepared using hybridoma technology, astaught by such references as: Kohler, et al., Nature 256:495 (1975); andHammerIing, et al., in: Monoclonal Antibodies and T-Cell HybridomasElsevier, N.Y., pp. 563-681 (1981). In general, this technology involvesimmunizing an immunocompetent animal, typically a mouse, with eitherintact chromosome 7p P-glycoprotein or a fragment derived therefrom.Splenocytes are then extracted from the immunized animal and are fusedwith suitable myeloma cells, such as SP₂O cells. Thereafter, theresulting hybridoma cells are selectively maintained in HAT medium andthen cloned by limited dilution (Wands, et al., Gastroenterology80:225-232 (1981)). Cells obtained through such selection are thenassayed to identify clones which secrete antibodies capable of bindingchromosome 7p P-glycoprotein.

Antibodies or fragments of antibodies of the invention may be used todetect the presence of chromosome 7p P-glycoprotein in any of a varietyof immunoassays. For example, antibodies may be used inradioimmunoassays or in immunometric assays, also known as “two-site” or“sandwich” assays (see Chard, “An Introduction to Radioimmune Assay andrelated Techniques,” in: Laboratory, Techniques in Biochemistry andMolecular Biology, North Holland Publishing Co., NY (1978)). In atypical immunometric assay, a quantity of unlabeled antibody is bound toa solid support that is insoluble in the fluid being tested, such asblood, lymph, cellular extracts and the like. Following the initialbinding of antigen to immobilized antibody, a quantity of detectablylabeled second antibody (which may or may not be the same as the first)is added to permit detection and/or quantitation of bound antigen (see,e.g. Radioimmune Assay Method, Kirkham, et al., Ed. pp. 199-206, E&SLivingstone, Edinburgh (1970)). Many variations of these types of assaysare known in the art and may be employed for the detection of 7pP-glycoprotein.

Antibodies to chromosome 7p P-glycoprotein may also be used inpurification procedures (see generally, Dean et al., AffinityChromatography, A Practical Approach, IRL Press (1986)). Typically,antibody is immobilized on a chromatographic matrix such as Sepharose,4B. The matrix is then packed into a column and the preparationcontaining chromosome 7p P-glycoprotein is passed through underconditions that promote binding, e.g., low salt conditions. The columnis then washed protein is eluted using buffer that promotes dissociationfrom antibody, e.g., buffer having an altered pH or salt concentration.The eluted protein may be transferred into a buffer, for example viadialysis, and thereafter either stored or used directly. Antibodies mayalso be used in Western blotting for the detection of chromosome 7pP-glycoprotein in a sample. For these types of assays, antibody may beused which has either been developed specifically to react withchromosome 7p P-glycoprotein or which reacts with an epitope of theprotein.

The detection of the chromosome 7p P-glycoprotein may be used todetermine whether tumor cells are multidrug resistant. Likewise,detection of changes in the expression of P-glycoprotein may be usefulin predicting the development of multidrug resistance in cells. The cDNAof this P-glycoprotein may be useful in designing primers for diagnosticPCR, probe design for diagnostic Northern blotting, RNase protectionassays, and for the design of antisense oligonucleotides complementaryto the predicted cDNA for use in gene-targeting strategies for thereversal of multidrug resistance. Both in vitro and in vivo diagnosticand therapeutic uses for antisense nucleotide sequences to thechromosome 7p P-glycoprotein are envisioned.

The primary amino acid sequence and protein structure of the chromosome7p P-glycoprotein may be utilized in the production of monoclonalantibodies (mAbs) that can be used in the diagnosis and therapy ofmultidrug resistant cancer. For example, synthetic peptides resemblingnative amino acid sequences from particular extracellular domains asdetermined by membrane topology prediction may be useful for developinginhibitory mAbs directed against extracellular epitopes of thechromosome 7p P-glycoprotein. Additionally, 10-20 mer synthetic peptidesequences derived from the primary amino acid sequence not included inthe above-mentioned extracellular loop sequences may be useful in thedevelopment of specific diagnostic monoclonal antibodies. Specific mAbsmay be employed in diagnostic FACS analysis, Western blotting, andimmunohistochemistry. Such mAbs may also be employed for in vivodiagnostic uses, where label-conjugated mAbs can be used to assess tumorburden, tumor localization or residual tumor mass following chemotherapyor surgical therapy of 7p 15-21 P-glycoprotein-expressing tumors.

Specific mAbs can also be used for therapeutic purposes in cancerpatients. In particular, they may be administered to reverse cancermultidrug resistance in patients receiving chemotherapeutic agents thatare substrates for 7p P-glycoprotein efflux, e.g., cisplatin. Inaddition, specific mAbs may be used therapeutically in cancer patientsfor tumor-specific cell killing, either administered in an unconjugatedform, resulting in immune-mediated tumor killing, or in a celltoxin-conjugated form (for example conjugated to radioactive iodine orchemical toxins), resulting in direct tumor-specific cell killing.

Specific mAbs can also be used for therapeutic purposes other thancancer multidrug resistance. Based on the predicted immunoregulatoryfunction of 7p P-glycoprotein, these mAbs can be given to patients toprevent and/or treat organ transplant rejection, and also diverseautoimmune diseases such as rheumatoid arthritis and multiple sclerosis.Furthermore, since P-glycoproteins function in the uptake, excretion andtissue-specific distribution of a variety of pharmacological andchemical compounds, and have been implicated in mechanisms of oralbioavailability, blood/brain barrier function and renal, hepatic andbiliary excretion mechanisms of several drugs, specific mAbs can beadministered therapeutically to alter the pharmacokinetics andavailability of those therapeutic drugs which are substrates for 7pP-glycoprotein-mediated transport function.

The compositions and methods of the present invention may have a numberof uses in addition to those described above. For example, pluripotentstem cells and tissue progenitor cells such as hematopoietic stem cells,neuroprogenitor cells and muscle progenitor cells are known to possessP-glycoprotein-like efflux activities for small molecules andfluorescent dyes. Chromosome 7p P-glycoprotein may play a role in thetransport of such substrates, and thus may serve as a marker for theisolation of such stem cells and progenitor cells via, for example, FACSanalysis. Also, since MDR1 P-glycoprotein appears to be involved incellular differentiation, cell proliferation, cell survival, and certainimmune responses, chromosome 7p P glycoprotein, due to its homology withMDR 1 P-glycoprotein, is expected to play a role in such physiologicalfunctions as well. Thus, chromosome 7p P-glycoprotein gene and proteinsequences may be useful in modulating pathophysiological disruptions ofthese MDR-related functions.

EXAMPLES

Since new genomic sequence information is currently being produced at arapid pace via the human genome project, databases containing suchgenomic information potentially contain sequences of heretoforeunidentified members of the P-glycoprotein family. MammalianP-glycoprotein family members share characteristic amino acid sequencesand protein epitopes, and assume similar conformations. Thus, a proteinhomology-based search was conducted in an attempt to identify novelP-glycoprotein-encoding genes. Gene-analytic and protein-analyticbioinformatics tools were utilized to further characterize the nucleicacid sequence and predicted protein structure of identified candidategenes. Specifically, the National Center for Biotechnology Information(NCBI) tblasn application was used to compare conserved amino acidsequences derived from the known structure of the human MDR1P-glycoprotein with the NCBI non-redundant homo sapiens nucleotidesequence database dynamically translated in all reading frames. Thesignature sequence common to members of the ABC transporter family, a 15mer amino acid sequence LSGGQKQRIAIARAL (SEQ ID NO: 17), was used toidentify human genomic DNA sequences encoding homologous proteinstructures. Known hexamer amino acid sequences of threeP-glycoprotein-specific monoclonal antibody (mAb)-binding epitopes werealso employed.

Human genomic DNA clones identified in the manner described above werescreened for vector contamination using the VecScreen program.Additionally, these clones were subjected to systematic homology mappingusing overlapping contiguous 20-mer amino acid sequences derived fromthe human MDR1 protein structure and the tblasn search program.Candidate genomic DNA sequences encoding homologous amino acid sequenceswere compared to open reading frame (ORF) sequences predicted in eachDNA clone using the NCBI ORF Finder program (Altschul, et al, NucleicAcids Res. 25:3389-402 (1997)). Genomic ORFs containing homologous DNAsequences were then analyzed using the NetGene2 software package inorder to predict intron splice sites in the candidate genes (Brunak etal., J. Mol. Biol. 220:49-65 (1991)).

A cDNA sequence was generated by conceptual linear transcription ofpredicted adjacent DNA exon structures. Utilizing this approach, twoadjacent overlapping human genomic clones, CTA-367017 (AC002486, 79611base pairs in length) and CTB-86D3 (AC005060, 120169 base pairs inlength, sequenced to the right) were identified as forming part of anunanchored island of unknown orientation on chromosome 7p15-21. Theseoverlapping clones were found to contain a gene sequence encoding anovel member of the human P-glycoprotein family.

In order to determine whether the predicted gene structure was expressedin human tissues, the generated cDNA sequence was compared to the humanNCBI dbest non-redundant expressed sequence tags (EST) database, asdescribed by Altschul et al., and several ESTs complementary topredicted exons from the genomic clone AC002486 were identified.Polymerase chain reaction (PCR) primers were then designed based onavailable sequence information in the database at the National Centerfor Biotechnology Information (NCBI) and the bioinfornatic analysis asdescribed above. Using these gene-specific oligonucleotide primers andthe PCR technique on reverse transcribed total messenger RNA (mRNA)isolated from several human cancer cell lines and normal human tissues,including the human G3361melanoma cell line, the MCF-7 breast carcinomacell line, the SCC25 squamous cell carcinoma cell line, the U937leukemia cell line, and normal peripheral: blood mononuclear cells(PBMC), cDNA sequences derived from the novel 7p15-21 P-glycoproteingene were amplified and the PCR products were subsequently sequencedusing the dideoxy chain termination method on both strands.

The intron-exon structure of several gene products encoded by the7p15-21 P-glycoprotein gene was determined by comparison of predictedand sequenced cDNA clones with genomic sequence information from the7p15-21 P-glycoprotein gene locus (clones AC002486 and AC005060), asshown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16. Protein structuresencoded by the new 7p15-21 gene were then generated by conceptual aminoacid translation of the predicted oligonucleotide sequences of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 and SEQ ID NO:16, as shown in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7and SEQ ID NO:8. These amino acid sequences were then compared with theNCBI non-redundant peptide sequence for sequence homology using the NCBIblastp program. The predicted amino acid sequences of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7and SEQ ID NO:8 were also classified using the PIR-International ProteinFamily Classification System (Barker, et al., Nucleic Acids Res. 28:41-4(2000); Huang et al., Nucleic Acids Res. 28:273-6 (2000)). Potentialfunctional characteristics of the predicted proteins were determined bycomparative analysis of the primary amino acid composition as well as byusing the TMHMM1.0 software package for the prediction of transmembranehelix formation in mammalian proteins (Sonnhammer et al., Ismb 6:175-82(1998)).

The novel 7p15-21 P-glycoprotein gene can encode several distinctP-glycoprotein isoforms which display 68% sequence homology with bothhuman MDR1 and MDR3. A similar degree of homology was found withrespective mouse and hamster isoforms of these human genes. Primaryamino acid sequence analysis suggests that the chromosome 7p15-21P-glycoprotein may express the C32 and anti-P-glycoprotein mAb bindingepitope, but not the C219 epitope conserved in all other knownP-glycoprotein isoforms (Georges, et al., Proc. Nad Acad Sci USA87:152-6 (1990)).

Structural prediction revealed that the 7p15-21 P-glycoprotein geneencodes P-glycoprotein isoforms which exhibit structural similaritiesbut also distinctive differences compared to known members of theP-glycoprotein family, as disclosed by Georges et al. For example, theprotein of SEQ ID NO:2 contains two ATP-binding domains which arelocated on opposite sides of the plasma membrane, providing for a uniqueextracellular ATP-binding domain which is predicted to bindextracellular ATP. Based on these distinctive differences, it ispredicted that 7p15-21 P-glycoprotein is not only involved in smallmolecule efflux, but that some of its isoforms are also functional inthe energy-dependent uptake of small molecules. The PIR classificationsystem confirmed the discovered chromosome 7p15-21 P-glycoprotein to bea member of the family of multidrug resistance proteins and the familyof ATP-binding cassette homology superfamilies.

PCR analysis using gene-specific primers demonstrated that cDNA encodingthe proteins of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 andSEQ ID NO:6, which in each case involves exons encoded on genomic cloneAC005060, was preferentially expressed in human melanoma cells but notmost other cancers tested, unlike cDNAs encoding the proteins of SEQ IDNO:7 and SEQ ID NO:8, which was found expressed in most cancers examinedand also physiologic human tissues. This emphasizes that a subset of 7p15-21 P-glycoprotein gene products can be selectively targeted incertain cancers that display particularly high degrees ofchemoresistance, such as human melanoma,

To assess the expression and function of 7p15-21 P-glycoprotein and theeffect of specific modulation on transport function and chemoresistance,polyclonal antibodies were raised against the MDR peptidesCGTSLILNGEPGYTI (SEQ ID NO:18) and RFGAYLIQAGRMTPEGC (SEQ ID NO:19),corresponding to distinct extracellular loop epitopes of 7p15-21P-glycoprotein, by injecting mice with these antigenic peptidesconjugated to the carrier substance KLH. To assess 7p15-21P-glycoprotein surface expression of human tumor cells, indirect surfaceimmunostaining and single color flow cytometry of freshly harvestedcells was performed. To assess the effects of 7p15-21 P-gp inhibition onP-gp-mediated fluorescent dye efflux, tumor cells were incubated withanti-7p15-21 P-glycoprotein polyclonal Ab followed by addition ofcalcein-AM and subsequent serial cell fluorescence measurements by flowcytometry.

These studies demonstrated that P-glycoprotein is expressed on tumorcells, and that the RFGAYLIQAGRMTPEGC (SEQ ID NO:19) epitope containedin the proteins of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5and SEQ ID NO:6, is preferentially expressed in human melanoma at highlevels, whereas the CGTSLILNGEPGYTI (SEQ ID NO:18) epitope, alsocontained in SEQ ID NO:7 and SEQ ID NO:8, is also expressed in othertypes of cancer and normal human cells. Antibodies against theCGTSLILNGEPGYTI (SEQ ID NO: 18) epitope inhibited both dye uptake andalso dye efflux dependent on cell type, indicating a dual function ofthe various gene products of 7p15-21 P-glycoprotein in these distinctprocesses. These antibodies also enhanced cell cytotoxicity ofcisplatinum in specific cell killing assays in melanoma and also breastcancer among others, indicative of their potential therapeuticusefulness in the treatment of cancer patients.

Certain cancers are known to exhibit chromosomal rearrangement in the7p15-21 region, and such mutations can be associated with the emergenceof the MDR phenotype. This raises the possibility that generearrangement in these cancers potentially results form episome anddouble minute (DM) chromosome formation during the process of geneamplification of 7p15-21 P-glycoprotein under mutagenic stresses such aschemotherapy. Cells expressing MDR1-mediated multidrug resistance areknown to undergo such chromosomal rearrangements and DM chromosomeformation (Scehoenlein et al., Mol. Biol. Cell 3:507-20 (1992); Mickleyet al., J. Clin. Invest. 99:1947-57 (1997); Knutsen et al., GenesChromosomes Cancer 23:44-54 (1998)). Thus, the chromosome 7p15-21P-glycoprotein gene products of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:5 and SEQ ID NO:6 may be selectively overexpressed in certaincancer cells, thereby contributing to the acquired drug resistance ofsuch cancer cells while remaining silent in normal cells. Thisdifferential expression pattern may be employed in the detection andreversal of multidrug resistance of tumorigenic mammalian cells.

1. A method of enriching stem cells from a cell mixture, comprisingcontacting a cell mixture with an antibody directed against a 7pP-glycoprotein and isolating stem cells bound to the antibody from thecell mixture.
 2. A method of obtaining a 7p P-glycoprotein positive stemcells comprising: obtaining a sample from a human donor; and isolating7p P-glycoprotein positive cells from the sample.
 3. The method of claim2, wherein the 7p P-glycoprotein positive cells are isolated usingimmobilized antibody against 7p P-glycoprotein.
 4. The method of claim3, wherein the 7p P-glycoprotein cells are isolated using antibody thathas been immobilized on beads.
 5. The method of claim 2, wherein the 7pP-glycoprotein cells are isolated using FACS analysis.
 6. A method ofcharacterizing a stem cell, comprising contacting a stem cell with anantibody directed against a 7p P-glycoprotein to determine if the stemcell is 7p P-glycoprotein positive.
 7. An isolated 7p P-glycoproteinpositive stem cell, wherein the stem cell has been isolated using anantibody directed against 7p P-glycoprotein.